This project is a major part of the Developmental Therapeutics Program drug discovery and development program within the Division of Cancer Treatment and Diagnosis. The emphasis is on preclinical drug discovery and preclinical drug development. In Vitro Cancer Cell Line Screen (IVCCLS) During FY2007, the cell-line screen added a 60thth tumor cell line, MDA-MB-468, for evaluation of the antitumor activity of both natural and synthetic agents. The screen includes cell lines that represent a variety of cancers, including leukemia, melanoma, and cancers of the lung, colon, brain, ovary, breast, prostate, and kidney. The aim is to prioritize, for further evaluation, synthetic compounds or natural product samples showing selective growth inhibition or cell killing of particular tumor cell lines. The primary screening paradigm was changed to incorporate an initial single drug-dose screen against all 60 cell lines in the screen. Drugs that show activity in the one-dose assay are retested in the standard assay that consists of a 5-concentration test using the 60 human tumor cell lines. The criteria used to identify active agents was developed based on historical data from the 5-dose screen and developed to establish a targeted pass rate of 2025% and to capture the majority of drugs that have previously been shown to be active in the standard screen. The concept of the one-dose screen is to more rapidly identify active drugs and eliminate inactive compounds so resources can be focused on the active compounds. The 60-cell line screen is unique in that the complexity of a 60-cell line dose response produced by a given compound results in a biological response pattern that can be utilized in pattern recognition algorithms. Using these programs, it is possible to assign a putative mechanism of action to a test compound, or to determine that the response pattern is unique and not similar to that of any of the standard prototype compounds included in the NCI database. In addition, following characterization of various cellular molecular targets in the cell lines, it is possible to select compounds most likely to interact with a specific target. In Vitro Molecular Targets Screening Program (MTSP) - The In Vitro Molecular Target Screening Program (MTSP) is a dedicated service providing direct support to the DCTD/DTP anticancer drug discovery program. This laboratory has developed the infrastructure and expertise for the operation of cell-free and cell-based high-throughput (HTS) molecular target-based assays. MTSP has developed the staff experience, instrumentation, and information technology infrastructure to carry out screening campaigns with large chemical libraries (>140,000 compounds). Several HTS screening campaigns have been completed and others are ongoing at the present time. In addition, MTSP has gained enhanced molecular biology experience by developing new methods for the transfection of human tumor cells with molecular target genes of interest and by optimizing expression systems for recombinant proteins to be utilized as reagents for our HTS molecular target screens. To complement these primary screens, the laboratory has developed assays for secondary testing of compounds showing activity in various HTS screens.